Enzymatic reactions exhibit remarkable selectivity and efficiency, the likes of which are rarely achieved in bench-top chemical reactions. While it is clear that the biochemical prowess of an enzyme arises from its highly-ordered structure, the detailed mechanism by which it functions has proven elusive. This is because enzymes are not simply static macromolecules that host an active site, as depicted by their crystal structure; rather, they are dynamic molecules whose choreographed motions can gate the transport of substrate to and from the active site and can modulate over time the activity of that site. To develop a mechanistic understanding into how enzymes function, it is essential to study this choreography of life with structurally-sensitive methods capable of ultrafast time resolution. In this report, we focus on time-resolved X-ray studies that employ the pump-probe method. Briefly, a laser pulse (pump) photoactivates or thermally excites a biomolecule, after which a suitably delayed X-ray pulse (probe) passes through the sample and records a diffraction or scattering pattern on a 2D detector. Thanks to significant capital equipment investments made by the NIH (over $1.2M since 2006), we have developed the ability to pursue studies of biomolecules on the BioCARS 14-IDB beamline via both time-resolved Laue crystallography and time-resolved SAXS/WAXS. Time-resolved Laue crystallography takes advantage of a polychromatic X-ray pulse, which can produce thousands of reflections in a single shot, and boosts substantially the rate at which time-resolved diffraction data can be acquired. The information needed to determine the protein's structure is encoded in the relative intensities of the diffraction spots observed. Since the structural information contained in a single Laue diffraction image is incomplete, repeated measurements at multiple crystal orientations are required to produce a complete set of data. Prior studies have generally required a small number of very large, homogeneous crystals, which has limited the application of this methodology to a handful of proteins. The impact of time-resolved Laue crystallography would be boosted significantly if we were to develop methods capable of acquiring high S/N diffraction images from a large number of relatively small crystals (30-35 microns), rather than small number of large crystals. To that end, we continue our efforts to develop novel microfluidic methods for growing crystals and delivering them in a fashion that can be automated. Briefly, we have developed an alternating drop microfluidic mixer that has been used to grow more than 1000 uniformly sized lysozyme crystals ( 30-35 microns) in a 1-m long glass capillary. A microfluidic crystal delivery system based on a home-built multi-axis syringe-pump tower is being developed with an aim to automate crystal delivery to the BioCARS 14-IDB beamline. This effort also includes the development of a high-speed diffractometer capable of rapidly and precisely positioning crystals at the intersection of the laser and X-ray beams. While much progress has been made, more work remains to be done. Our aim to automate the acquisition of X-ray diffraction images from thousands of crystals without user intervention will hopefully be realized within the coming year. Time-resolved Laue crystallography, as its name implies, can only be performed on crystalline samples. The intermolecular forces that maintain crystalline order constrain large amplitude conformational motion, and this loss of flexibility may perturb or even inhibit the function of a protein. Though Laue crystallography stands alone in its ability to track structural changes in proteins on ultrafast time scales with near-atomic spatial resolution, it is crucial to also study structural dynamics of biomolecules in solution where the full range of conformational motion is permitted. Without external alignment forces, biomolecules in solution are randomly oriented, and the structural information contained in their orientationally-averaged diffuse scattering pattern is one dimensional. Nevertheless, it is well known that the SAXS region of the diffraction pattern reports on the size and shape of the biomolecule, while the WAXS region is sensitive to secondary and tertiary structure. Time-resolved SAXS/WAXS scattering patterns therefore provide 'fingerprints' that can be correlated with protein structure via molecular models, and can assess which models best describe reaction pathways in solution. Our time-resolved SAXS/WAXS diffractometer currently employs a secondary K-B mirror pair to focus the X-ray beam onto the sample capillary with independent control of the the vertical and horizontal dimensions, a very small beamstop (0.51 mm diameter), and a large area (340x340 mm), high-speed (up to 10 Hz) X-ray detector. With the sample-detector distance set at 185.8 mm, scattering data can be acquired over a broad range of q (momentum transfer) spanning 0.02 to 5.4 inverse Angstroms, which corresponds to spatial resolution below 1.2 Angstroms. To mitigate the adverse effects of radiation damage during X-ray exposure, the capillary containing the protein solution is rapidly translated over a 20-mm span using a home-built, high-speed diffractometer that is based on 1-micron resolution linear motor translation stages capable of more than 1-g acceleration. Thanks to a closed-loop circulation system, about 150 microliter of protein solution is sufficient to acquire a high signal-to-noise ratio data set. With our recently improved capillary holder and high-precision temperature controller, we are able to characterize structural changes over a broad range of temperatures spanning from approximately -16 to 120 degrees Celsius. Moreover, thanks to the relatively small spot size that can be generated with the secondary K-B mirror pair, it is possible to focus a 1 mJ infrared pulse down to a dimension small enough to heat samples in a glass capillary by more than 20 degrees Celsius. When setting the sample temperature just below its unfolding temperature, this magnitude T-jump is sufficient to trigger unfolding of the biomolecule, and allows us to investigate the dynamics of protein unfolding with unprecedented spatial resolution. The time-resolution achieved is currently limited by the duration of the infrared laser pulse, which is about 5 ns. Prior time-resolved studies of the R to T structure transition in human hemoglobin unveiled complicated kinetics which seemed to point to heterogeneity in the R state. To explore this possibility, we pursued a time-resolved T-jump study of the R state of human hemoglobin. If more than one R state exists, one would expect their relative population to be temperature dependent, and would lead to a time-dependent change in its X-ray scattering pattern as the relative population of two (or more) states comes into equilibrium at the higher temperature. Indeed, we recently reported in Dynamics of Quaternary Structure Transitions in R-State Carbonmonoxyhemoglobin Unveiled in Time-Resolved X-ray Scattering Patterns Following a Temperature Jump, J Phys Chem B 122, 11488-11496, that human hemoglobin has two (or more) R states, whose rate of interconversion is approximately 30 microseconds. Armed with this knowledge, we are working to develop a comprehensive model capable of unveiling in mechanistic detail how this allosteric transition enables hemoglobin to efficiently transport oxygen from the lungs to the tissues. As our time-resolved SAXS/WAXS methodology becomes more precise and easier to use, we expect it to become an ever more important complement to time-resolved Laue studies and time-resolved optical spectroscopy studies of biomolecules, and will help provide a structural basis for understanding how biomolecules function.